Reoviruses are prototypical members of the Reoviridae family of segmented dsRNA viruses that include important human (rotavirus) and veterinary (blue tongue disease) pathogens. The mechanisms by which 10 unique positive-sense viral RNAs are localized to viral factories for packaging into progeny core particles is poorly understood and will be addressed by the experiments in this proposal. The paths taken by reovirus positive-sense RNAs after they are transcribed will be examined by both in situ hybridization and immunostaining of bromouridine labeled viral positive-sense RNAs in infected cells. The mechanism for localization of viral RNAs to viral factories will be examined by microinjection studies of viral or control RNAs into reovirus infected cells, or transfected cells expressing reovirus RNA-binding proteins. If viral RNAs are specifically localized to viral factories, trans- and cis-acting factors involved in localization will be identified. Mechanisms for non-specific localization of RNA to factories will also be examined. Understanding the RNA localization process may lead to the development of new anti-viral strategies, and will aid in the development of a robust reverse genetics system for dsRNA viruses.